Irene Martín

Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures

A rapid real-time polymerase chain reaction (PCR) technique using SYBR Green detection system, has been developed for the quantification of red deer, fallow deer, and roe deer DNAs in meat mixtures. The method combines the use of cervid-specific primers that amplify a 134, 169, and 120bp of the 12S rRNA gene fragment of red deer, fallow deer and roe deer, respectively, and universal primers that amplify a 140bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The C(t) (threshold cycle) values obtained with the 18S rRNA primers are used to normalize those obtained from each of the cervid-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat treated binary mixtures of red deer, fallow deer or roe deer meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target cervid DNAs in the range 0.1-0.8%, depending on the species and treatment of the meat samples analyzed.

Differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes.

This work describes the differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR targeting sequences from two molecular markers: the mitochondrial displacement loop (D-loop) region and the nuclear melanocortin receptor 1 (MC1R) gene. A polymorphic D-loop fragment (∼270bp) was amplified and sequenced in a number of wild and domestic Sus scrofa meat samples, to find a nucleotide region suitable for PCR-RFLP analysis. Sequence data showed the presence of only a few point mutations across Sus scrofa D-loop sequences, not allowing direct discrimination between wild boar and domestic swine meats. Later, the MC1R gene was targeted and Sus scrofa-specific primers designed to amplify a 795bp MC1R fragment. Subsequent RFLP analysis of the MC1R swine-specific amplicons allowed selection of BspHI and BstUI endonucleases to carry out intraspecific Sus scrofa differentiation. Digestion of MC1R amplicons with the chosen enzymes generated characteristic PCR-RFLP profiles that allowed discrimination among meats from wild and domestic swine specimens. The technique also enabled the detection of samples that yielded heterozygous profiles, suggesting hybrids resulting from wild boar and domestic pig breeding. The PCR-RFLP reported here, targeting the MC1R gene may be routinely applied to verify the correct labelling of game products.

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs.

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.

Species-specific PCR for the identification of ruminant species in feedstuffs.

A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA mitochondrial gene has been developed for the specific identification of bovine, ovine and caprine DNAs in feedstuffs. The primers designed generated specific fragments of 84, 121 and 122pb length for bovine, ovine and caprine species, respectively. The specificity of the primers designed was tested against 30 animal species including mammals, birds and fish, as well as eight plant species. Analysis of experimental feedstuffs demonstrated that 0.1% of raw and heated bovine, ovine or caprine tissues can be easily detected using the species-specific primers developed. The performance of this method is not affected by prolonged heat treatment, and consequently it could be very useful to verify the origin of the raw materials in products submitted to denaturing technologies, for which other methods cannot be applied.

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene.

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.

PCR identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) targeting specific sequences from the mitochondrial D-loop region.

A polymerase chain reaction (PCR) assay was developed for the identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by using oligonucleotides targeting mitochondrial D-loop sequences. A D-loop region (∼700-1000 bp) was firstly amplified and sequenced from various game and domestic meat DNAs, and three primer sets were then designed on the basis of nucleotide multialignment of the generated D-loop sequences. As expected from sequence analysis, PCR amplification of the targeted D-loop fragments was successfully achieved from chamois (88 bp), pyrenean ibex (178 bp), and mouflon (155 bp) meats, showing adequate specificity and reproducibility against a number of game and domestic meats. Mouflon and sheep meats were amplified together in accordance to the high nucleotide identity of their mt D-loop sequences. In this work, satisfactory amplification was also accomplished in the analysis of experimentally pasteurized (72°C for 30min) and sterilized (121°C for 20min) meats, with a detection limit of ∼0.1% for each of the targeted species. The proposed PCR assay represents a rapid and straightforward method for the detection of possible adulterations in game meat products.

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.

Identification of raw and heat-processed meats from game bird species by polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial D-loop region

Polymerase chain reaction-RFLP analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), chukar partridge (Alectoris chukar), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), and woodpigeon (Columba palumbus). Polymerase chain reaction amplification was carried out using a set of primers flanking a conserved region of approximately 310 bp from the mitochondrial D-loop region. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of HinfI, MboII, and Hpy188III endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. Consistent results were obtained with both raw and heat-processed meats.

Real-Time PCR for Quantitative Detection of Bovine Tissues in Food and Feed

A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues-oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133 degrees C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.